A methodology for detection and quantitation of protein phosphotyrosine (tyr-P) was developed. The method is rapid (sample preparation and assay is about 4 hours) and highly sensitive (detection limit: 100-500 pmol). With 32p labeled proteins, detection can be achieved with trace quantities. The method was used to survey phosphoproteins for tyr-P content. No tyr-P was detected with histone samples and casein. Moreover, when these proteins were phosphorylated with cAMP-dependent protein kinase and casein kinase, no tyr-P detected. Egg phosphoproteins, phosvitin and vitellin, were shown to contain tyr-P. This is the first identification of phosphotyrosine proteins from notransformed cells. Antisera to tyr-P was produced. The IgG fraction which bound tyr-P was purified by affinity chromatography. The phosphotyrosine antibodies showed high affinity for tyr-P and exhibited low cross reactivity with tyrosine, serine phosphate, theronine phosphate and inorganic phosphate. A 32P labeled phosphotyrosyl protein was produced for assaying phosphotyrosyl protein phosphatase(s) in cells. The substrate was produced by deadenosylation of (32P)-adenylylated glutamine synthetase. The phosphotyrosyl protein was a substrate for several phosphatases. Detection of phosphotyrosyl protein phosphastase activites in normal and transformed cells is currently being studied.